Use of a 2-hydroxy-4-trifluoromethylbenzoic acid derivatives as inhibitors of the activation of the nuclear transcription factor NF-kappaB

ABSTRACT

Neurodegenerative diseases, such as dementia, including Alzheimer&#39;s disease, can be treated or prevented by administering an effective amount of triflusal or its metabolite, or a pharmaceutically acceptable salt or a prodrug thereof.

[0001] This application is a continuation of U.S. Ser. No. 09/701,270which was a filing under 35 U.S.C. 371 of PCT/ES99/00154, filed May 26,1999.

FIELD OF THE INVENTION

[0002] The present invention relates to inhibitors of the nucleartranscription factor kappaB (NF-κB) and to their use in therapy.Specifically, the present invention relates to the use of2-hydroxy-4-trifluoromethylbenzoic acid derivatives to inhibit theactivation of the transcription factor NF-κB.

DESCRIPTION OF THE PRIOR ART

[0003] The control of the expression of proteins plays a key role bothin the maintenance of the normal function of cells and hence oforganisms, as well as in the development of pathological processes. Thiscontrol is effected through the so-called transcription factors. One ofthese factors is the group of proteins known as nuclear transcriptionfactor NF-κB, formed by a family of intimately related dimericcomplexes. NF-κB exists in an inactive form in the cytoplasm of manytypes of cells. In response to a stimulus, it becomes activated and isthen translocated to the nucleus, where it binds to DNA and regulatesthe transcription of various genes. The activation of NF-κB can beinduced by several agents such as infalmmatory cytokines (for example,tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (1L-1β)),mitogens, bacterial lipopolysaccharides (LPS), viruses, oxidants (forexample, H₂O₂ and ozone), phorbol esters and ultraviolet light. Amongthe various genes whose expression is regulated by NF-κB, many genesinvolved in immune and inflammatory responses are included. Thus, amongothes, NF-κB regulates the expression of proinflammatory cytokines suchas IL-1β, Interleukin-2 (IL-2), interleukin-6 (IL-6), TNF-α andgranulocyte-macrophage colony stimulating factor (GM-CSF); chemokinessuch as interleukin-8 (IL-8), RANGES, macrophage inflammatory protein-1α(MIP-1α), monocyte chemotactic protein-1 (MCP-1) and eotaxin;inflammatory enzymes such as inducible nitric oxide synthase (iNOS),cyclooxygernase-2 (COX-2), 5-lipoxygenase (5-LO) and cytosolicphospholipase A₂ (cPLA₂); adhesion molecules such as intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)and E-selectin; and receptors such as the interleukin-2 receptor and theT-cell receptor (P. J. Barnes and I. M. Adcock, Trends Pharmacol. Sci.1997, 18, 46-50).

[0004] Dysfunctions in the activation of NF-κB and its dependent geneshave been associated with several pathologies such as acuteinflammation, septic shock, transplant rejection, radiation damage,ischemia and reperfusion damage and neurodegenerative diseases (P. A.Baeuerle and T. Henkel, Annu. Rev. Immunol. 1994, 12, 141-179), asthmaand other chronic inflammatory diseases (P. J. Barnes and I. M. Adcock,Trends Pharmacol. Sci. 1997, 18, 46-50), osteoporosis (Y. Abu-Amer andM. Mehrad Tondravi, Nature Med. 1997, 3(11), 1189-1190), and cancer (M.A. Sovak et al., J. Clin. invest. 1997, 100 (12), 2952-2960). Moreover,elevated levels of NF-κB have been detected in synovial tissue ofpatients with rheumatoid arthritis (H. Asahara et al., Biochem. Mol.Biol. Int., 1995, 37(5), 827-32), in central nervous system samples ofmultiple sclerosis patients (D. Gveric et al., J. Neuropathol. Exp.Neurol. 1998, 57(2), 168-78) and in samples of atherosclerotic tissue(K. Brand et al., J. Clin. Invest. 1996, 97(7), 1715-22), and it hasbeen described that amyloid β peptide, which accumulates in plaques ofAlzheimer patients, activates NF-κB in central nervous system cells (C.Behl et al., Cell 1994, 77, 817-827). A high increase in the nucleartranslocation of NF-κB has also been observed in dopaminergic neurons ofpatients with Parkinson's disease (S. Hunot et al., Proc. Natl. Acad.Sci. USA 1997, 94(14), 7531-7536). Furthermore, NF-κB has also beenreported to be involved in the transcriptional activation of virusessuch as human immunodeficiency virus (HIV), cytomegaloviruses,adenoviruses and herpesviruses.

[0005] On the other hand, it has been shown that the cytokines,inflammatory enzymes, adhesion molecules and other proteins whoseexpression is regulated by NF-κB play an important role in a broad rangeof disorders such as inflammation; asthma; adult respiratory distresssyndrome (ARDS); immunoinflammatory and autoimmune diseases such asrheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory boweldisease, lupus and glomerulonephritis; arthrosis; septic shock;atherosclerosis; cancer; osteoporosis; preterm labour; transplantrejection; neurodegenerative diseases such as dementia, includingAlzheimer's disease, Parkinson's disease and amyotrophic lateralsclerosis; and viral infections.

[0006] In view of the above, the agents which are able to modulate theactivity of the transcription factor NF-κB and/or the expression ofgenes which are dependent on this transcription factor could be of greatutility as therapeutic agents for the treatment or prevention of theabove-mentioned disorders. It is thus of great interest to find agentswhich are capable of regulating NF-κKB activity.

[0007] 2-Acetyloxy-4-trifluoromethylbenzoic acid, better known by itsInternational Nonproprietary Name (INN) triflusal, is a plateletaggregation inhibitor marketed for the treatment of thromboembolicdiseases under the trademark Disgren®. Its main metabolite,2-hydroxy-4-trifluoromethylbenzoic acid (also known by the acronym HTB),also possesses a remarkable activity as platelet antiaggregant. Bothcompounds are described in U.S. Pat. No. 4,096,252.

[0008] The present inventors have found that, surprisingly, bothtriflusal and its metabolite, HTB, inhibit NF-κB activation. Moreover,it has been found that both compounds are potent inhibitors of theexpression of genes that are transcriptionally regulated by NF-κB. Dueto this new activity now discovered, triflusal and HTB are potentiallyuseful in the treatment or prevention of disorders where the activationof NF-κB and its dependent genes is involved, such as those mentionedabove.

DESCRIPTION OF THE INVENTION

[0009] The present invention is based upon the finding that triflusaland its metabolite, HTB, are potent inhibitors of the activation of thetranscription factor NF-κB. As mentioned above, NF-κB is an ubiquitoustranscription factor that acts by binding to DNA, activating in thismanner the expression of various genes, many of them involved in theimmune and inflammatory response. The present invention shows thattriflusal and HTB inhibit the activation of NF-κB induced by variousagents such as TNF-α, immune complexes and LPS in several types ofcells, such as human umbilical vein endothelial cells (HUVEC),macrophages and monocytes. Moreover, it is also shown that triflusal andHTB inhibit the expression of several proteins in whose transcriptionalregulation NF-κB is involved, such as for example VCAM-1, iNOS, COX-2,MCP-1 and TNF-α. Therefore, triflusal and HTB are useful as therapeuticor preventive agents in those pathological situations where NF-κB and/orthe proteins whose expression is regulated by this transcription factorare involved.

[0010] Triflusal and HTB can be generically represented by means offormula I:

[0011] wherein R represents hydrogen (HTB) or COCH₃ (triflusal).

[0012] It is an object of the present invention to provide the use of acompound of formula I for the manufacture of a medicament useful forinhibiting the activation of the transcription factor NF-κB. The use ofa pharmaceutically acceptable salt of a compound of formula I or of aprodrug thereof are also encompassed within the scope of the presentinvention.

[0013] Another object of the present invention is to provide the use ofa compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of genes which are dependent on and/orregulated by, at least partially, the transcription factor NF-κB. In apreferred embodiment, the gene encodes IL-1β, IL-2, IL-6, TNF-α, GM-CSF,IL-8, RANTES, MIP-1α, MCP-1, eotaxin, iNOS, COX-2, 5-LO, cPLA₂, ICAM-1,VCAM-1, E-selectin, IL-2 receptor or T-cell receptor, and morepreferably encodes VCAM-1, iNOS, COX-2, MCP-1 or TNF-α.

[0014] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of disorders associated with the activation of thetranscription factor NF-κB and/or the expression of genes which aredependent on this transcription factor. In a preferred embodiment, thedisorder is inflammation; asthma; adult respiratory distress syndrome(ARDS); immunoinflammatory and autoimmune diseases such as rheumatoidarthritis and other arthritic conditions, multiple sclerosis, psoriasis,inflammatory bowel disease, lupus and glomerulonephritis; arthrosis;septic shock; atherosclerosis; cancer; osteoporosis; preterm labour;transplant rejection; neurodegenerative diseases such as dementia,including Alzheimer's disease, Parkinson's disease and amyotrophiclateral sclerosis; and viral infections.

[0015] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of COX-2.

[0016] Another object of the present invention is to provide the use ofa compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of diseases mediated by COX-2. In a preferred embodiment, thedisease mediated by COX-2 is rheumatoid arthritis and other arthriticconditions, arthrosis, preterm labour, dementia or cancer.

[0017] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of VCAM-1.

[0018] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of diseases mediated by VCAM-1. In a preferred embodiment,the disease mediated by VCAM-1 is atherosclerosis, rheumatoid arthritis,lupus, multiple sclerosis, inflammatory bowel disease, asthma, allergicrhinitis and tumor metastasis.

[0019] Another object of the present invention is to provide the use ofa compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of iNOS.

[0020] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of diseases mediated by iNOS. In a preferred embodiment, thedisease mediated by iNOS is inflammation, septic shock, inflammatorybowel disease and neurodegenerative diseases.

[0021] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of TNF-α.

[0022] Another object of the present invention is to provide the use ofa compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of diseases mediated by TNF-α. In a preferred embodiment, thedisease mediated by TNF-α is rheumatoid arthritis, rheumatoidspondylitis, gouty arthritis and other arthritic conditions, arthrosis,sepsis, septic shock, endotoxic shock, toxic shock syndrome, adultrespiratory distress syndrome, cerebral malaria, chronic pulmonaryinflammatory disease, silicosis, pulmonary sarcoidosis, pulmonaryfibrosis, hepatitis, osteoporosis and other bone resorption diseases,reperfusion injury, transplant rejection, multiple sclerosis, lupus,fever and myalgias due to infections, cachexia, acquired immunedeficiency syndrome (AIDS), inflammatory bowel disease and pyresis.

[0023] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament useful forinhibiting the expression of MCP-1.

[0024] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of diseases mediated by MCP-1. In a preferred embodiment, thedisease mediated by MCP-1 is atherosclerosis, glomerulonephritis,rheumatoid arthritis, pulmonary fibrosis, restenosis, asthma, psoriasis,inflammatory bowel disease, multiple sclerosis and transplant rejection.

[0025] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of neurodegenerative diseases, particularly dementia,Parkinson's disease and amyotrophic lateral sclerosis.

[0026] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of immunoinflammatory and autoimmune diseases, preferablyrheumatoid arthritis and other arthritic conditions, multiple sclerosis,psoriasis, inflammatory bowel disease, lupus and glomerulonephritis.

[0027] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of arthrosis.

[0028] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of cancer.

[0029] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of atherosclerosis.

[0030] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the preventionof preterm labour.

[0031] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of inflammation.

[0032] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of asthma or adult respiratory distress syndrome.

[0033] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of septic shock.

[0034] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of osteoporosis.

[0035] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of viral infections.

[0036] It is also an object of the present invention to provide the useof a compound of formula I or a pharmaceutically acceptable salt or aprodrug thereof for the manufacture of a medicament for the treatment orprevention of transplant rejection.

[0037] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the activation of the transcription factor NF-κB.

[0038] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of genes which are dependent on and/orregulated by, at least partially, the transcription factor NF-κB.

[0039] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of disorders associated with the activationof the transcription factor NF-κB and/or the expression of genes whichare dependent on this transcription factor.

[0040] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of COX-2.

[0041] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of diseases mediated by COX-2, preferablyrheumatoid arthritis and other arthritic conditions, arthrosis, pretermlabour, dementia or cancer.

[0042] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of VCAM-1.

[0043] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of diseases mediated by VCAM-1, preferablyatherosclerosis, rheumatoid arthritis, lupus, multiple sclerosis,inflammatory bowel disease, asthma, allergic rhinitis and tumormetastasis.

[0044] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of iNOS.

[0045] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of diseases mediated by iNOS, preferablyinflammation, septic shock, inflammatory bowel disease andneurodegenerative diseases.

[0046] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of TNF-α.

[0047] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of diseases mediated by TNF-α, preferablyrheumatoid arthritis, rheumatoid spondylitis, gouty arthritis and otherarthritic conditions, arthrosis, sepsis, septic shock, endotoxic shock,toxic shock syndrome, adult respiratory distress syndrome, cerebralmalaria, chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, pulmonary fibrosis, hepatitis, osteoporosis and other boneresorption diseases, reperfusion injury, transplant rejection, multiplesclerosis, lupus, fever and myalgias due to infections, cachexia,acquired immune deficiency syndrome (AIDS), inflammatory bowel diseaseand pyresis.

[0048] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forinhibiting the expression of MCP-1.

[0049] The present invention also provides the use of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof forthe treatment or prevention of diseases mediated by MCP-1, preferablyatherosclerosis, glomerulonephrits, rheumatoid arthritis, pulmonaryfibrosis, restenosis, asthma, psoriasis, inflammatory bowel disease,multiple sclerosis and transplant rejection.

[0050] The present invention also provides a method for inhibiting theactivation of the transcription factor NF-κB in a mammal in needthereof, which comprises administering to said mammal a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt or a prodrug thereof. The mammal is preferably a humanbeing.

[0051] The present invention also provides a method for inhibiting theexpression of genes which are dependent on and/or regulated by, at leastpartially, the transcription factor NF-κB in a mammal in need thereof,which comprises administering to said mammal a therapeutically effectiveamount of a compound of formula I or a pharmaceutically acceptable saltor a prodrug thereof.

[0052] The present invention also provides a method for the treatment orprevention of disorders associated with the activation of thetranscription factor NF-κB and/or the expression of genes which aredependent on this transcription factor in a mammal in need thereof,which comprises administering to said mammal a therapeutically effectiveamount of a compound of formula I or a pharmaceutically acceptable saltor a prodrug thereof.

[0053] The present invention also provides a method for inhibiting theexpression of COX-2 in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0054] The present invention also provides a method for the treatment orprevention of diseases mediated by COX-2, preferably rheumatoidarthritis and other arthritic conditions, arthrosis, preterm labour,dementia or cancer, in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0055] The present invention also provides a method for inhibiting theexpression of VCAM-1 in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0056] The present invention also provides a method for the treatment orprevention of diseases mediated by VCAM-1, preferably atherosclerosis,rheumatoid arthritis, lupus, multiple sclerosis, inflammatory boweldisease, asthma, allergic rhinitis and tumor metastasis, in a mammal inneed thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt or a prodrug thereof.

[0057] The present invention also provides a method for inhibiting theexpression of iNOS in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0058] The present invention also provides a method for the treatment orprevention of diseases mediated by iNOS, preferably inflammation, septicshock, inflammatory bowel disease and neurodegenerative diseases, in amammal in need thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt or a prodrug thereof.

[0059] The present invention also provides a method for inhibiting theexpression of TNF-α in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0060] The present invention also provides a method for the treatment orprevention of diseases mediated by TNF-α, preferably rheumatoidarthritis, rheumatoid spondylitis, gouty arthritis and other arthriticconditions, arthrosis, sepsis, septic shock, endotoxic shock, toxicshock syndrome, adult respiratory distress syndrome, cerebral malaria,chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, pulmonary fibrosis, hepatitis, osteoporosis and other boneresorption diseases, reperfusion injury, transplant rejection, multiplesclerosis, lupus, fever and myalgias due to infections, cachexia,acquired immune deficiency syndrome (AIDS), inflammatory bowel diseaseand pyresis, in a mammal in need thereof, which comprises administeringto said mammal a therapeutically effective amount of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof.

[0061] The present invention also provides a method for inhibiting theexpression of MCP-1 in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or a prodrugthereof.

[0062] The present invention also provides a method for the treatment orprevention of diseases mediated by MCP-1, preferably atherosclerosis,glomerulonephritis, rheumatoid arthritis, pulmonary fibrosis,restenosis, asthma, psoriasis, inflammatory bowel disease, multiplesclerosis and transplant rejection, in a mammal in need thereof, whichcomprises administering to said mammal a therapeutically effectiveamount of a compound of formula I or a pharmaceutically acceptable saltor a prodrug thereof.

[0063] It is also encompassed within the scope of the present inventiona pharmaceutical composition which comprises a therapeutically effectiveamount of a compound of formula I or a pharmaceutically acceptable saltor a prodrug thereof for the treatment or prevention of disordersassociated with the activation of the transcription factor NF-κB and/orthe expression of genes which are dependent on this transcriptionfactor, and more preferably for the treatment or prevention ofinflammation; asthma; adult respiratory distress syndrome (ARDS);immunoinflammatory and autoimmune diseases such as rheumatoid arthritisand other arthritic conditions, multiple sclerosis, psoriasis,inflammatory bowel disease, lupus and glomerulonephritis; arthrosis;septic shock; atherosclerosis; cancer; osteoporosis; preterm labour;transplant rejection; neurodegenerative diseases such as dementia,including Alzheimer's disease, Parkinson's disease and amyotrophiclateral sclerosis; and viral infections.

[0064] The pharmaceutically acceptable salts of a compound of formula Iinclude any of the salts commonly used in pharmaceutical chemistry, suchas for example the salts with inorganic cations such as sodium,potassium, calcium, magnesium, lithium, aluminium, zinc, etc as well asthe salts formed with ammonia and other pharmaceutically acceptableamines.

[0065] Throughout the present description, the term prodrug of acompound of formula I means any precursor compound of a compound offormula I that is capable of being metabolized and release in vivo acompound of formula I, that is triflusal or HTB.

[0066] By NF-κB it is to be understood any member of the family ofproteins known by this name.

[0067] By gene which is dependent on and/or regulated by, at leastpartially, the transcription factor NF-κB it is to be understood anygene having in its promoter region one or more NF-κB binding sites. Thelist of genes regulated by NF-κB mentioned above under the heading“Description of the prior art” is cited only as an example and it is notto be understood as limiting the scope of the invention in any way.

[0068] By disorder associated with the activation of the transcriptionfactor NF-κB and/or the expression of genes which are dependent on thistranscription factor it is to be understood any disease or pathologicalstate where the activation of NF-κB and/or the proteins whose expression(i.e. the expression of the gene that encodes them) is regulated by thistranscription factor are involved, at least partially. The lists ofthese diseases mentioned above are cited only as examples and thus arenot to be understood as limiting the scope of the present invention inany case.

[0069] The term neurodegenerative diseases includes, among others,dementias, such as Alzheimer's disease; diseases involving movementdysfunction, such as Parkinson's disease; progressive ataxias; andamyotrophies of neuronal origin, such as amyotrophic lateral sclerosis.

[0070] By the term dementia it is to be understood any pathologycharacterized by an impairment of the cognitive functions, such as forexample Alzheimer's disease, post-traumatic dementia or dementiafollowing infection as well as mixed situations.

[0071] The term inflammatory bowel disease includes both ulcerativecolitis and Crohn's disease as well as any other type of variant ofinflammatory bowel disease.

[0072] The term transplant rejection refers both to tissue transplantrejection, such as for example graft-versus-host disease, as well asorgan transplant rejection.

[0073] Processes for preparing triflusal or HTB are disclosed in theabove-mentioned U.S. patent (U.S. Pat. No. 4,096,252).

[0074] As mentioned above, the compounds of formula I inhibit theactivation of the transcription factor NF-κB and therefore can be usedto inhibit said activation in mammals, preferably in human beings. Thedose of a compound of formula I necessary to modulate the activation ofthe transcription factor NF-κB, or any other use herein described, willdepend upon the disorder to be treated, the severity of the symptoms,the age and body weight of the patient as well as the chosen route ofadministration. Any person skilled in the art will be able to readilydetermine the appropriate doses depending on these factors withouthaving to incur in undue experimentation. In human therapy, doses willgenerally be in the range between about 30 mg and about 3000 mg daily ofa compound of formula I, which can be administered in one or severaldosage units. Depending on the particular disease to be treated and thepatient's situation, however, doses outside this range might be needed,which, as mentioned above, may be readily determined by those skilled inthe art without requiring undue experimentation.

[0075] The compounds of formula I can be administered in the form of anypharmaceutical formulation, the nature of which will depend, as it iswell known, upon the route of administration and the nature of thedisease to be treated. These pharmaceutical compositions can be preparedby conventional methods, using compatible, pharmaceutically-acceptableexcipients or vehicles. Examples of such compositions include capsules,tablets, syrups, powders and granulates for the preparation ofextemporaneous solutions, injectable preparations, etc. A preferredroute of administration for the compounds of formula I is by the oralroute. For example, they can be administered as hard gelatine capsulescontaining for example 50, 100, 200, 300, 400 or 500 mg of a compound offormula I or a pharmaceutically acceptable salt or a prodrug thereof.

BRIEF EXPLANATION OF THE FIGURES

[0076]FIG. 1 shows the inhibitory effect of triflusal and HTB on theactivation of the transcription factor NF-κB induced by TNF-α in humanumbilical vein endothelial cell (HUVEC) cultures.

[0077]FIG. 2 shows the inhibitory effect of triflusal and HTB on theactivation of the transcription factor NF-κB induced by immune complexes(IC) in rat macrophages.

[0078]FIG. 3 (A and B) shows the inhibitory effect of HTB on theactivation of the transcription factor NF-κB induced by bacteriallipopolysaccharide (LPS) in human peripheral blood mononuclear cells.

[0079]FIG. 4 shows the inhibitory effect of triflusal and HTB on theexpression of VCAM-1 mRNA induced by TNF-α in HUVEC.

[0080]FIG. 5 shows the inhibitory effect of HTB (5A) and triflusal (5B)on the production of nitrite induced by immune complexes in ratmacrophages.

[0081]FIG. 6 shows the effect of triflusal and HTB on LPS-induced COX-2in human mononuclear cells: (6A) inhibitory effect of triflusal on COX-2expression; (6B) inhibitory effect of HTB on COX-2 expression; (6C)inhibition of prostaglandin E₂ (PGE₂) production elicited by triflusal;(6D) inhibition of PGE₂ production elicited by HTB.

[0082]FIG. 7 shows the inhibitory effect of triflusal administeredorally on COX-2 expression (7A) and on PGE₂ production (7B) in acarrageenan-induced inflammation model in the rat.

[0083]FIG. 8 shows the inhibitory effect of HTB on MCP-1 expressioninduced by immune complexes (IC) in human monocytic cell line THP-1.

[0084]FIG. 9 shows the inhibitory effect of triflusal and HTB on TNF-αexpression induced by bacterial lipopolysaccharide (LPS) in humanperipheral blood mononuclear cells.

[0085] The following examples illustrate the utility of triflusal andHTB as inhibitors of the activation of NF-κB and its dependent genes. Inany case are they to be construed as limiting the scope of the presentinvention. The following abreviations have been used in the examples:

[0086] EDTA: ethylenediaminetetraacetic acid

[0087] DTT: 1,4-dithiothreitol bp: base pairs

[0088] PBS: phosphate-buffered saline

[0089] RT-PCR: reverse transcriptase polymerase chain reaction

[0090] dNTP: deoxyribonucleoside triphosphate

[0091] DNA: deoxyribonucleic acid

[0092] RNA: ribonucleic acid

[0093] MTT: thiazolyl blue

[0094] TBS: Tris-buffered saline

[0095] ATP: adenosyl triphosphate

[0096] DMSO: dimethylsulfoxide

[0097] FCS: fetal calf serum

EXAMPLE 1 Inhibition of the Activation of NF-κB Induced by TNF-α inHUVEC

[0098] A. Cell Culture

[0099] Human umbilical vein endothelial cells (HUVEC) were obtained bythe procedure of Dejana et al. (J. Cell Biol 1987, 104(5), 1403-1411) bytreatment of the umbilical vein with 0.2% collagenase P from C.histolyticum (Boehringer Manheim GmbH, Manheim, Germany) for 20 minutesat 37° C. Next, cells were cultured in M199 medium (Flow Lab, Herts,U.K.) containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/mlamphotericin B and 20% fetal calf serum. Primary cultures were plated in25 cm² plastic flasks. After 24 hours cells were washed to remove thosecells which had not adhered to the flask surface. Then, the same mediumcontaining 10% fetal calf serum, 50 μg/ml endothelial cell growthsupplement factor and 100 μg/ml heparin was added. After culturing for5-7 days, cells reached confluence and were detached from the flasksurface with 0.05% trypsin and 0.02% EDTA (Flow Lab). The reaction wasinhibited by the addition of fetal calf serum, and then cells werewashed and plated again in culture medium. Cells were grown toconfluence in gelatin-coated flasks. Cells used for the experiments werefrom passages 2-7.

[0100] B. Treatment of HUVEC Cells with TNF-α and ElectrophoreticMobility Shift Assay (EMSA).

[0101] In this experiment, HUVEC cells were preincubated with Triflusaland HTB, at concentrations in both cases of 2 and 4 mM. Then these cellswere stimulated with 100 U/ml TNF-α (Genzyme Diagnostics, Cambridge,Mass. USA) for 90 minutes. Next, HUVEC cells were washed with coldhypotonic lysis buffer (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mMMgCl₂, 0.5 mM DTT (1,4-dithiothreitol), 0.5 mM phenylmethylsulfonylfluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptine and 0.6% Nonidet P-40)and were kept on ice for 10 minutes. Then, they were vortexed vigorouslyfor 10 seconds. Unbroken cells were eliminated by centrifugation at1,000× g for 10 minutes. The nuclei were collected by centrifugation at15,000× g for 1 minute in a microcentrifuge. The nuclear pellet wasresuspended in a high salt extraction buffer (25% glycerol and 0.5 MKCl). The nuclear extract was obtained by centrifugation for 30 minutesat 105,000× g in a Optima TI ultracentrifuge (Beckmann) using a TLA100.2 rotor. A 22 bp double-stranded oligonucleotide containing NF-κBsequences was used as probe. This probe was end-labeled with (γ-³²P)ATPusing T4 polynucleotide kinase and was purified by minicolumnchromatography. The κB sequence used was, 5′-AGTTCAGGGGMTTTCCCAGGC-3′and the complementary 5′-GCCTGGGAAATTCCCCTGMCT-3′. 10 μg of the purifiednuclear protein was incubated for 20 minutes on ice with theradiolabeled oligonucleotide probe (2-6×10⁴ cpm) in 25 μl of reactionbuffer consisting of 2 μg poly(dI-dC), 10 mM Tris HCl pH 7.5, 100 mMNaCl, 1 mM EDTA, 1 mM DTT, 8% Ficoll and 4% glycerol.Nucleoprotein-oligonucleotide complexes were resolved by electrophoresisin a nondenaturing polyacrylamide gel in Tris-borate/EDTA buffer for 3hours at 175 V and at 4° C. The gel was dried and autoradiographed withan intensifying screen at −80° C. and for 2 to 12 hours. The specificityof the DNA(probe)protein complex was confirmed by competition of the³²P-labeled probe with a 300-fold excess of unlabeled probe, whichshowed no presence of the labeled probe in the DNA-protein complex (datanot shown). The lane labeled as control corresponds to cells incubatedfor 90 minutes in the absence of TNF-α.

[0102] C. Results

[0103] The results of this experiment are shown in FIG. 1. Bothtriflusal and HTB concentration-dependently inhibit the activation ofNF-κB induced by TNF-α in HUVEC.

EXAMPLE 2 Inhibition of the Activation of NF-κB Induced by ImmuneComplexes (IC) in Rat Macrophages

[0104] A. Isolation and Culture of Rat Peritoneal Macrophages

[0105] Rat peritoneal cavity cells were extracted and resuspended inDMEM culture medium in the absence of serum previously supplemented with100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin, 2 mMglutamine and 0.5 mM L-arginine. Cells were incubated for 2 hours inculture plates at 37° C. and those which had not adhered to the plateswere removed by washing three times with the same fresh medium. Morethan 95% of the adherent cells were macrophages, as assessed by theirability to engulf zymosan particles and nonspecific esterase staining.Macrophages were kept at 37° C. under a 5% CO₂ atmosphere and two hourslater non-adherent cells were removed and the peritoneal macrophagesadhered to the plates were incubated with 100 μg/ml IgG/ovalbumin immunecomplexes prepared from rabbit antiserum for 2 hours in the presence orabsence (vehicle) of triflusal or HTB (4 mM, both).

[0106] B. Electrophoretic Mobility Shift Assay (EMSA).

[0107] After incubation, macrophages were washed twice with PBS and thedegree of activation (binding to DNA) of the transcription factor NF-κBwas determined using the EMSA assay, described in detail in Example 1.

[0108] C. Results

[0109]FIG. 2 shows the results obtained with triflusal and HTB in thisexperiment. A representative example of the obtained in two independentexperiments is shown. Both triflusal and HTB markedly inhibit theactivation of NF-κB induced by immune complexes in rat macrophages.

EXAMPLE 3 Inhibition of the Activation of NF-κB Induced by BacterialLipopolysaccharide (LPS) in Human Peripheral Blood Mononuclear Cells(PBMC)

[0110] A. Isolation and Culture of Human Mononuclear Cells.

[0111] Mononuclear cells (PBMC) were obtained from blood of healthydonors from the Hospital de Sant Pau (Barcelona) who had not taken anyantiinflammatory drug during a period of not less than 2 weeks beforeblood extraction. Starting from a volume of about 80 ml of heparinizedhuman blood (10 U/ml), this was diluted (1:1) with PBS (pH 7.4;Dulbecco) without calcium, magnesium nor sodium bicarbonate. 15 ml ofFicoll solution (d=1.077 at 20° C.; Biochrom KG) were placed in 50-mlFalcon tubes. To this solution a volume of about 25 ml per tube of theblood previously diluted with PBS was carefully added and the tubes werethen centrifuged at 1,200 g for 20 minutes. Mononuclear cellsconcentrate on a whitish interphase between the plasma and the Ficollsolution. This interphase was collected with a Pasteur pipette and wasdiluted 1:1 with PBS. It was then centrifuged at 300 g for 10 min. Theresulting pellet was resuspended in 50 ml PBS and was again centrifugedat 200 g for 10 min, in order to remove platelet contamination. Finally,the resulting pellet was resuspended in 20 ml of RPMI-1640 culturemedium (GibcoBRL) supplemented with 10% fetal calf serum.

[0112] Isolated PBMCs were analyzed by standard Wright-Giemsa stainingto examine if the cells displayed the morphological features of viablemononuclear cells and determine the different types of cells isolated(an average of 90% lymphocytes and 10% monocytes). Prior to the varioustreatments, cell viability was determined by the Trypan Blue exclusionassay. Cells were diluted at a concentration of 2.5 million/ml with RPMImedium containing 10% fetal calf serum and were incubated (37° C., 5%CO₂) 1 hour in 6-well plates (2 ml/well, 5 million PBMC) without addingany drug (control) or adding HTB (1-3 mM). Next, cells were incubatedfor further 10 minutes in the presence of 10 μg/ml E.colilipopolysaccharide (LPS, 026:B6 serotype; Sigma). After incubation, asample (100 μl) was taken in order to carry out a cell count as well ascell viability controls by measuring the ability of mitochondrialdehydrogenases to convert the soluble tetrazolium salt, MTT, into theinsoluble product formazan (MTT assay). Cells were also incubated withHTB (3 mM) in the absence of LPS. In all cases, cell viability was equalto or higher than 95%.

[0113] B. Electrophoretic Mobility Shift Assay (EMSA).

[0114] After incubation, PBMCs were washed twice with PBS and the degreeof activation (binding to DNA) of the transcription factor NF-κB wasdetermined using the EMSA assay, described in detail in Example 1.

[0115] C. Results

[0116]FIG. 3 shows the results obtained with HTB in this experiment. Arepresentative example of the obtained in two independent experiments isshown. HTB markedly inhibits the activation of NF-κB induced by LPS inhuman PBMCs.

EXAMPLE 4 Inhibition of the Expression of VCAM-1 in HUVEC Induced byTNF-α

[0117] A. Synthesis of First Strand cDNA and PCR of VCAM-1

[0118] Primers used for the detection of VCAM-1 mRNA by RT-PCR weredesigned from the human gene sequence (EMBL/GenBank AC: M30257), usingthe Wisconsin Package Version 9.1, Genetics Computer Group (GCG),Madison, Wis. Their sequences were 5′-TGTCACTGTAAGCTGCAAG-3′ and5′-TTCCAGCCTGGTTAATTC-3′, corresponding to nucleotides 1090-1108 and1589-1572 (L. Osborn et al., Cell 1989, 59(6), 1203-1211). Total RNA wasextracted from cultured cells using the guanidium isothiocyanate method(P. Chomczynski and N. Sacchi, Anal Biochem. 1987, 162(1), 156-159).cDNA first strand was synthesized from total RNA by reversetranscription reaction. The reaction was carried out using 0.2 mg/mltotal RNA (preheated at 68° C. for 10 minutes), 2.5 μl H₂O, 20 U ofRNAase inhibitor, 4 μl buffer 5×, 2 μl 0.1 M DTT, 4 μl 2.5 mM dNTP, 1 μl0.1 mM hexanucleotides and 200 U of Moloney-murine leukemia virusreverse transcriptase. The reaction was carried out at 37° C., for 60minutes in a volume of 20 μl. VCAM-1 cDNA was amplified by PCR in areaction mixture containing 2 μl DNA, 10 μl H₂O, 2.5 μl buffer 10×, 0.75μl 50 mM MgCl₂, 1.0 μl 2.5 mM dNTP, 1.25 μl of each primer (sense andantisense) and 0.25 μl Taq DNA polymerase 5 U/ml. A negative controlusing water was included in each PCR reaction. The amplificationconditions were as follows: an initial cycle of denaturation at 94° C.for 5 minutes, and then 30 cycles comprising: denaturation at 94° C. for30 seconds, primer annealing at 59° C. for 30 seconds, and extension at72° C. for 1 minute; and finally a cycle of extension at 72° C. for 7minutes. The relative amounts of each amplified cDNA were determined bymeasuring the density of the bands stained with ethidium bromide usingthe Gel Doc documentation system and the Molecular Analyst software fromBio-Rad Laboratories, Hercules, Calif. The expression of β-actin wasused as control for assaying the expression of a constitutivelyexpressed gene.

[0119] Specifically, the effect of Triflusal (4 mM) and HTB (4 mM) onthe regulation of the expression of VCAM-1 mRNA induced by TNF-α (100U/ml) in HUVEC was studied. Thus, cells were incubated in the presenceor absence of TNF-α and with Triflusal and HTB. After one hour, totalRNA was extracted and subjected to the reverse transcription reaction.Then, an amplification reaction by PCR was carried out with the primersdesigned for the sequences of the molecules VCAM-1 and human β-actin.The PCR products were separated by electrophoresis in a 1.8% agarose geland were later quantified. The molecular weight of the amplificationproducts was determined from the electrophoretic migration of DNA sizestandards.

[0120] B. Results

[0121] The results obtained in this experiment are shown in FIG. 4. Bothtriflusal and HTB at a concentration of 4 mM completely inhibit theexpression of VCAM-1 mRNA induced by TNF-α in HUVEC. The absence ofeffect on the expression of β-actin mRNA shows the selectivity of thetested compounds for the transcription factor NF-κB.

EXAMPLE 5 Inhibition of the Expression of iNOS in Rat PeritonealMacrophages Induced by Immune Complexes

[0122] A. Determination of the Production of NO by Rat PeritonealMacrophages.

[0123] Rat peritoneal cells were obtained and resuspended in DMEMculture medium in the absence of serum and supplemented withantibiotics. Macrophages were isolated by their ability to adhere toculture plates following incubation for 2 hours at 37° C. Non-adherentcells were removed and it was then checked that more than 95% of theadherent cells were macrophages, as assessed by their ability to engulfzymosan particles and nonspecific esterase staining. Culture plates werekept at 37° C. under a 5% CO₂ atmosphere, and the peritoneal macrophagesadhered to the plates were incubated with 100 μg/ml IgG/ovalbumin immunecomplexes in the presence or absence (Control) of triflusal or HTB(0.1-20 mM, both). Drugs were added 10 minutes before the addition ofIgG/ovalbumin and the production of NO was determined as the nitritepresent after 24 hours.

[0124] B. Determination of NO and Nitrite.

[0125] iNOS expression was indirectly measured as the production of NO.NO released from macrophage cultures was determined indirectly by theaccumulation of nitrites. To one milliliter of cell culture (0.5 millioncells in medium without phenol red) was added 100 μl of a solution of 1mM sulphanilic acid and 100 mM HCl (final concentration). Afterincubation for five minutes, the medium was aspirated and centrifuged inan eppendorf microcentrifuge. 50 μl naphthylenediamine (1 mM finalconcentration) were added and after 15 minutes of incubation theabsorbance of the sample was measured at 548 nm and was compared with aNaNO₂ standard. The production of NO was expressed as nmol NO₂ ⁻/mgprotein.

[0126] C. Results

[0127] The results obtained in this experiment are shown in FIG. 5A forHTB and in FIG. 5B for triflusal. Points represent the mean ±standarderror of the mean (SEM) from 7 to 9 experiments, each performed induplicate. IC₅₀ values calculated for triflusal and HTB from thecorresponding graphs were 1.13±0.12 and 1.84±0.34 mM, respectively.

[0128] Similar results were obtained when macrophages were incubatedwith LPS instead of immune complexes.

EXAMPLE 6 Inhibition of COX-2 Expression Induced by BacterialLipopolysaccharide (LPS) in Human Peripheral Blood Mononuclear Cells(PBMC)

[0129] A. Isolation and Culture of Human Mononuclear Cells.

[0130] Mononuclear cells (PBMC) were obtained from blood of healthydonors from the Hospital de Sant Pau (Barcelona) who had not taken anyantiinflammatory drug during a period of not less than 2 weeks beforeblood extraction. Starting from a volume of about 80 ml of heparinizedhuman blood (10 U/ml), this was diluted (1:1) with PBS (pH 7.4;Dulbecco) without calcium, magnesium nor sodium bicarbonate. 15 ml ofFicoll solution (d=1.077 at 20° C.; Biochrom KG) were placed in 50-mlFalcon tubes. To this solution a volume of about 25 ml per tube of theblood previously diluted with PBS was carefully added and the tubes werethen centrifuged at 1,200 g for 20 minutes. Mononuclear cellsconcentrate on a whitish interphase between the plasma and the Ficollsolution. This interphase was collected with a Pasteur pipette and wasdiluted 1:1 with PBS. It was then centrifuged at 300 g, 10 min. Theresulting pellet was resuspended in 50 ml PBS and was again centrifugedat 200 g for 10 min in order to remove platelet contamination. Finally,the resulting pellet was resuspended in 20 ml RPMI-1640 culture medium(GibcoBRL) supplemented with 10% fetal calf serum.

[0131] Isolated PBMCs were analyzed by standard Wright-Giemsa stainingto examine if the cells displayed the morphological features of viablemononuclear cells and determine the different types of cells isolated(an average of 90% lymphocytes and 10% monocytes). Cells were diluted ata concentration of 2.5 million/ml with RPMI medium containing 10% fetalcalf serum and were incubated (37° C., 5% CO₂) 19 hours in 6-well plates(2 ml/well, 5 million PBMC). Incubations were carried out in thepresence of 10 μg/ml E. coli lipopolysaccharide (LPS, 026:B6 serotype;Sigma) without adding any drug (control) or adding Triflusal or HTB(0.1-5 mM). Prior to incubations, cell viability controls were performedusing the Trypan Blue exclusion assay. After incubation, a sample (100μl) was taken in order to perform a cell count as well as cell viabilitycontrols by measuring the ability of mitochondrial dehydrogenases toconvert the soluble tetrazolium salt, MTT, into the insoluble productformazan (MTT assay). Cells were also incubated with HTB and Triflusalat the same concentrations but without adding LPS. In all cases, cellviability was equal to or higher than 95% both at the beginning of theexperiment and after 19 hours incubation.

[0132] B. Immunoblot Assays

[0133] After incubation, cells were centrifuged for 5 min at 1,000 g.The supernatant was collected and stored at −70° C. for laterdetermination of PGE₂ levels as a measure of COX-2 activity, and thepelleted cells were resuspended in 5 ml PBS and centrifuged again (5min, 1,000 g). The resulting pellet was resuspended in 50 μl cell lysisbuffer (PBS with 1% Nonidet-40 and 1 mM EDTA) and incubated in ice for15 minutes. The resulting mixture was centrifuged at 20,000 g for 15 minand the supernatant was collected. 5 μl of the supernatant were takenand diluted 1/20 with PBS in order to determine the concentration ofprotein using the BCA Protein Assay Reagent (Pierce).

[0134] The remaining supernatant was then mixed in a 1:1 ratio withelectrophoresis gel loading buffer (50 mM Tris; SDS, 2% w/v; glycerol,10% v/v; β-mercaptoethanol, 50 μl/ml and bromophenol blue, 2 mg/ml) andboiled for 5 min. Samples were centrifuged at 10,000 g for 2 min andthen subjected to discontinuous electrophoresis in SDS-Polyacrylamidegel (4% stacking gel/7.5% separating gel) at a variable intensity and afixed voltage of 200V, until the front was only a few millimeters fromthe gel end (about 1 hour).

[0135] Proteins were transferred to a nitrocellulose membrane, using acooled TE 22 Mighty Small Transfer Unit (Hoefer) system, at a voltage of100 V for 2 hours. When the transfer was finished, membranes werestirred overnight at 4° C. in blocking buffer (1:4 dried fat-free milkin TBS containing 0.1% Tween 20).

[0136] Blocked membranes were incubated 1 hour under stirring with agoat polyclonal antibody raised against human COX-2 (Santa CruzBiotechnology, Inc.), and after washing were incubated 1 hour with ahorseradish peroxidase-labeled antibody (Rabbit Anti-goatIgG-Horseradish Peroxidase, Immunopure, Pierce) and the antibody boundto the protein was visualized by chemoluminescence (ECL, Amershan).

[0137] Finally, the supernatants from each experiment that had beenstored at −70° C. were defrozen and the amount of PGE₂ in solution wasdetermined using specific ELISA kits (Amershan-Biotrak RPN22).

[0138] C. Results

[0139] The results obtained in this experiment are shown in FIG. 6. Theresults shown correspond to two representative immunoblots from theobtained in five independent experiments (FIG. 6A: triflusal; FIG. 6B:HTB) and the mean ±SEM of the quantification of PGE₂ in the supernatantsof the cultures corresponding to said experiments (FIG. 6C: triflusal;FIG. 6D: HTB). Both triflusal and HTB concentration-dependently inhibitCOX-2 expression as well as PGE₂ production.

EXAMPLE 7 Inhibition of COX-2 Expression in Rat Inflammatory ExudateCells

[0140] A. General Method

[0141] Lewis rats (175-200 g) were used in this study. Rats wererandomly distributed in groups of 5 animals. An air pouch was producedby subcutaneous injection of 20 ml of sterile air into the intrascapulararea of animals in each group. Every two days, 10 ml of air wereinjected again into the cavity to keep the space open. Seven days afterthe first injection of air, 2 ml of a 1% carrageenan solution wasinjected into the air pouch in all groups to produce an inflammatoryreaction. Test compound was administered orally 30 min beforecarrageenan administration. The animals were killed 6 h later and thevolume of exudate was measured. The type and number of cells presentwere determined using standard Wright-Giemsa staining and a CoulterCounter cell counter, respectively. The exudate was centrifuged at 400 gat 4° C. for 7 min and PGE₂ concentration was determined byenzyme-immunoassay (Amershan-Biotrak RPN222). The cell pellet wasresuspended in 2 ml cold 0.85% NaCl. To eliminate red cellcontamination, cells were subjected to a selective cellular lysis by theaddition of 6 ml cold water for 20 seconds. The isotonicity of thecellular suspension was restored by the addition of 2 ml 3.5% NaCl.Finally, the cellular suspension was centrifuged under the sameconditions mentioned above and the pellet was resuspended in lysisbuffer at a density of 2×10⁸ cells for immunoblot assays (see Example6).

[0142] B. Results

[0143] The results are shown in FIG. 7. FIG. 7A corresponds to arepresentative immunoblot and FIG. 7B shows the mean ±SEM of thequantification of PGE₂ in the rat exudate (n=4). The oral administrationof triflusal (3-30 mg/kg) dose-dependently inhibits COX-2 expression inthe cells present in the exudate as well as PGE₂ production.

EXAMPLE 8 Inhibition of the Expression of Monocyte Chemotactic Protein-1(MCP-1) Induced by Immune Complexes in the Human Monocytic Cell LineTHP-1

[0144] A. Cell Culture And Determination of MCP-1 Levels

[0145] Human monocytic THP-1 cells (3×10⁶ cells/well) were cultured inplastic dishes in RPMI 1640 culture medium supplemented with penicillin(100 U/ml), streptomycin (100 μg/ml), gentamycin (50 μg/ml), glutamine(2 mM) and 2% heat-inactivated fetal calf serum. Cells were cultured inthe presence of HTB (2 and 4 mM) or vehicle and were activated with 100μg/ml immune complex aggregates (A-IgG). The production of soluble MCP-1was determined by ELISA using a commercially available kit (R&D SystemsInc.; Minneapolis, Minn.). The detection limit of the system was 5μg/ml.

[0146] B. Results

[0147] The results obtained in this assay are shown in FIG. 8. HTB, bothat a concentration of 4 mM and 2 mM, causes a complete inhibition ofMCP-1 expression induced by immune complexes in THP-1.

[0148] EXAMPLE 9

Inhibition of the Expression of TNF-α Induced by BacterialLigopolysaccharide (LPS) in Human Peripheral Blood Mononuclear Cells(PBMC)

[0149] A. Isolation and Culture of Human Mononuclear Cells.

[0150] Mononuclear cells (PBMC) were obtained from blood of healthydonors from the Hospital de Sant Pau (Barcelona) following the proceduredescribed in example 3. Cells were diluted at a concentration of 2million/ml in RPMI medium supplemented with 10% fetal calf serum andwere incubated (37° C., 5% CO₂) with triflusal, HTB or vehicle (DMSO) inthe presence of 10 μg/ml E. coli lipopolysaccharide (LPS, 026:B6serotype; Sigma) for 19 hours. The cell suspension was then centrifugedat 2,000 g for 10 minutes at 4° C., and the resulting supernatant wasstored at −70° C. for later analysis. The cytokine content wasdetermined by enzymatic immunoassay, after 1/100 dilution of thesamples.

[0151] B. Results

[0152] The results obtained with triflusal and HTB in this experimentare shown in FIG. 9. Both triflusal and HTB (1 and 0.3 mM) almostcompletely inhibit the LPS-induced TNF-α production. The results areexpressed as the mean ±standard error of the mean from 2-5 separateexperiments, each performed in triplicate.

EXAMPLE 10 Inhibition of the Activation of NF-κB in Post-Natal LongEvans Black-Hooded Rat Glial Cells

[0153] A. General Method

[0154] This study was carried out using post-natal (P9) Long Evansblack-hooded rats. Each group consisted of 6 animals subjected to anexperimental lesion, plus two control animals of the same age. Theexperimental lesion was induced by intracortical injection (sensorimotorarea) of N-methyl-D-aspartate (NMDA), which causes a marked localneuronal degeneration. Triflusal (30 mg/kg) was administered orally inthree doses (from day 7 to 9) every 24 hours. Glial reactivity wasinduced by NMDA injection at postnatal day 9, one hour after the lasttriflusal dose. At different times (2-24 h) after this last dose,animals were killed, brains were extracted and cut in a cryostat andsections were processed using immunocytochemical and histochemicaltechniques to determine NF-κB activation in microglia and astrogliausing double staining: NF-κB-lectin and NF-κB-GFPA. In parallel, sliceswere cut on a vibratome to determine the degree of microglial andastroglial reactivity by histoenzymatic techniques (B. Castellano etal., J. Histochem. Cytochem., 1991, 39(5), 561-568).

[0155] B. Results

[0156] In control animals, cortical neurons but not glial cells showedconstitutively activated NF-κB. This basal activation is inhibited bypretreatment with triflusal. In those animals where an excitotoxiclesion was performed with NMDA, a rapid activation of NF-κB was observedin glial cells. Pretreatment with triflusal at 30 mg/kg p.o. completelyinhibited NF-κB activation, both in astroglial and microglial cells.

EXAMPLE 11 Triflusal Prevents Neuronal Cell Death in Cocultures ofNeurons and Astrocytes Induced by Oxygen/Glucose Deprivation (OGD)

[0157]A. General Method

[0158] To carry out this study, an in vitro model of neuronal ischemiabased on cocultures of neurons and glial cells was used. Primarycultures of type 1 astrocytes were prepared from 1-day old Wistar rats.Astrocytes were plated onto 60 mm, poly-D-lysine-coated plates. Thesecells were allowed to grow until they were confluent (about 11 days) andthen rat primary neurons were plated onto them and were allowed to growfor 10 days. In addition, separate cultures of each one of the two typesof cells were prepared.

[0159] Half of the cultures were exposed for four hours tooxygen-glucose deprivation (OGD), followed by a 24-hour recovery period.Both the cells exposed to OGD and control cells were treated at thestart of OGD with 0, 10 and 30 μg/ml triflusal in a series ofexperiments and with 0, 20 and 100 μg/ml HTB in another series. Afterthe 24-hour recovery period, the release of lactate dehydrogenase (LDH)into the medium was determined as a measure of cellular death, as wellas the degree of apoptosis in the cultures (using the TUNEL assay) andtotal neuron and astrocyte counts present in the coculture (usingHoescht staining).

[0160] B. Results

[0161] In the cultures exposed to OGD a marked increase in LDH releaseas well as in the number of apoptotic neurons was observed, as comparedto controls. The various concentrations of triflusal or HTB tested inthis study completely inhibited both effects. Therefore, in this modelboth triflusal and its metabolite HTB can prevent apoptosis and neuronaldegeneration induced by oxygen and glucose deprivation.

EXAMPLE 12 Inhibition of Adjuvant-Induced Arthritis in the Rat

[0162] A. Arthritis Induction

[0163] Adjuvant-induced arthritis is characterized by the development,from day 14 after adjuvant injection, of a chronic inflammation ofimmunological origin in several joints, with accumulation ofinflammatory cells and release of cytokines.

[0164] For this study, male Lewis rats with body weight between 100 and150 g were used. Before the start of the study animals were acclimatedfor a period of at least 5 days. Animals were fasted for 18 hours beforethe study, with water ad libitum.

[0165] Throughout the study, animals were allowed free access todrinking water, except during observation periods.

[0166] Groups of five animals were randomized (Sham, Control andTriflusal). The duration of the study was 28 days. Arthritis was inducedon day I of the study by subplantar administration of 0.1 ml of anemulsion formed with 10 mg M. butyricum and 10 ml Freund's incompleteadjuvant (Difco) to the right hindpaw of the animals from the Controland Triflusal groups. Sham animals received 0.1 ml Freund's incompleteadjuvant. Triflusal was administered daily from day 1 of the study at adose of 10 mg/kg p.o. in Tween 80 (1%). On day 28 of the development ofarthritis, the volume of the contralateral paw to that receiving theadjuvant injection was determined using a UGO BASILE 7150plethysmometer. The inhibition of the increase in volume was calculatedas follows:

% Inh.=100−((T−S)/(C−S))*100

[0167] Where: T=Triflusal group; C=Control group; and S=Sham group

[0168] B. Results

[0169] Oral administration of triflusal for 28 days at the dose of 10mg/kg produced a 63.1±8.0% inhibition of the increase in volume ofimmunological origin induced by M. butyricum and adjuvant in controlanimals.

EXAMPLE 13 Study of the Viability of Various Cell Lines AfterAdministration of HTB

[0170] A. General Method

[0171] Several cell lines obtained from the American Type CultureCollection (ATCC) were cultured at 37° C. and under a 5% CO₂ atmosphere.Each cell line was grown in an appropriate culture medium and within theexponential phase (Table I). TABLE I CELL LINE CULTURE MEDIUM U-937 RPMI1640 + 10% FCS (human histiocytic lymphoma) 143.98.2 DMEM + 10% FCS(human osteosarcoma) 1321N1 DMEM + 5% FCS + 0.5% Penicillin- (humanastrocytoma) Streptomycin Jurkat RPMI 1640 + 10% FCS (human acute T cellleukemia) COLO 205 RPMI 1640 + 10% FCS (human colon adenocarcinoma)

[0172] For the cell viability studies 24-well plates were used, where0.5×10⁶ cells/ml (for 24-hour studies), 0.25×10⁶ cells/ml (for 48-hourstudies) or 0.125×10⁶ cells/ml (for 72-hour studies) were incubated.Next, different concentrations of HTB (1-3 mM) were added and cells wereincubated for different time periods (37° C., 5% CO₂). After incubation,the supernatant from each well was extracted, and cells were washed withculture medium without fetal calf serum. Then, the supernatant wasextracted and 200 μl culture medium without fetal calf serum were addedto each well. In addition, 20 μl substrate were added to determine cellviability. This method is based on the ability of viable cells totransform the colourless substrate into a coloured substance that isexcreted to the supernatant (EZ4U, Biomedica Gmbh.). After incubatingcells for 1 hour at 37° C. and 5% CO₂, 200 μl supernatant were collectedand the absorbance was measured at 450 nm. The absorbance was alsodetermined at 620 nm as a measure of the existing non-specific value.

[0173] B. Results

[0174] The results of cell viability determinations are shown on TableII. Incubation with HTB leads to the cell death of the various tumoralcell lines tested. This cell death is concentration- and time-dependent.

[0175] Table II.—Percentage of Cell Viability. TABLE II Percentage ofcell viability. 0 2 hours hours 5 hours 24 hours 48 hours 72 hours U-9371 mM HTB 100 100 98.8 74.4 39.6 4.8 3 mM HTB 100 96.1 92.6 13.9 7.5 N.D.JURKAT 1 mM HTB 100 100 76.8 11.3 3.6 1.6 3 mM HTB 100 100 62.6 6.2 5.4N.D. 1321N1 1 mM HTB 100 99.7 94.2 87.5 67.6 N.D. 3 mM HTB 100 85.3 67.262.2 17.8 N.D. COLO 205 1 mM HTB 100 100 87.2 84.8 37.4 7.6 3 mM HTB 10094.5 69.2 39.6 14.3 N.D. 143.98.2 1 mM HTB 100 100 100 93.9 83.2 50.4  3mM HTB 100 98.1 82.7 52.2 16.1 N.D.

[0176] N.D.: not determined

[0177] The results from the assays described in examples 1, 2 and 3 showthat triflusal and HTB inhibit the activation of the transcriptionfactor NF-κB. They also show that this inhibition is independent of theinducing agent and the type of cell. These results show the utility oftriflusal and HTB in the treatment or prevention of those disorderswhere NF-κB is involved.

[0178] The results from example 4 show that triflusal and HTB inhibitVCAM-1 expression. It has been described that the VCAM-1 gene has NF-κBbinding sites (C. Weber et al., Arterioscler. Thromb. 1994, 14(10),1665-1673). It has been shown that adhesion molecules such as VCAM-1 areinvolved in disorders such as atherosclerosis (K. D. O'Brien et al., J.Clin. Invest. 1993, 92, 945-951), rheumatoid arthritis, lupus, multiplesclerosis, inflammatory bowel disease, asthma, allergic rhinitis andtumor metastasis. By inhibiting NF-κB activation and VCAM-1 expression,both triflusal and HTB may be of particular utility in the treatment orprevention of VCAM-1-mediated disorders such as all those mentionedabove and specially atherosclerosis.

[0179] In example 5 it is shown that triflusal and HTB also inhibit iNOSexpression, which is regulated at transcriptional level, at leastpartially, by NF-κB (U. Förstermann et al., Biochem. Pharmacol. 1995,50(9), 1321-1332). It has been shown that iNOS is involved inpathologies such as inflammation, septic shock, inflammatory boweldisease and neurodegenerative diseases such as dementia and Parkinson'sdisease (J. E. Ogden and P. K. Moore, Trends Biotechnol. 1995, 13(2),70-78). By inhibiting NF-κB activation and iNOS expression, triflusaland HTB may be of particular utility in the treatment or prevention ofiNOS-mediated disorders and specially inflammation, septic shock,inflammatory bowel disease and neurodegenerative diseases such asdementia and Parkinson's disease.

[0180] The results of examples 6 and 7 show that triflusal and HTBinhibit COX-2 expression both in vitro and in vivo. It has beendescribed that the gene encoding COX-2 has NF-κB binding sites (S. B.Appleby et al., Biochem. J. 1994, 302, 723-727). COX-2 has beenassociated with pathologies such as rheumatoid arthritis and otherarthritic conditions, arthrosis, preterm labour, dementia, particularlyAlzheimer's disease (T. A. Sandson and O. Felician, Exp. Opin. Invest.Drugs 1998, 7(4), 519-526), and cancer (M. Oshima et al., Cell 1996,87(5), 803-809; K. Subbaramaiah et al., Cancer Res. 1996, 56(19),4424-4429). By inhibiting NF-κB activation and COX-2 expression,triflusal and HTB may be of particular utility in the treatment orprevention of COX-2-mediated disorders and specially rheumatoidarthritis and other arthritic conditions, arthrosis, preterm labour,dementia and cancer.

[0181] The results from example 8 show that HTB inhibits also MCP-1expression, which is regulated at transcriptional level, at leastpartially, by NF-κB (T. Martin et al., Eur. J. Immunol. 1997, 27(5),1091-1097). It has been described that an excessive or unregulated MCP-1production is involved in disorders such as glomerulonephritis (B. H.Rovin et al., Lab. Invest 1994, 71(4), 536-542), rheumatoid arthritis(P. M. Villiger et al., J. Immunol. 1992, 149(2), 722-727), pulmonaryfibrosis (H. N. Antoniades et al., Proc. Natl. Acad. Sci. USA 1992,89(12), 5371-5375), restenosis, asthma, psoriasis, inflammatory boweldisease, multiple sclerosis and transplant rejection, and it is the mostpotent chemotactic factor detected in macrophage-rich atheroscleroticplaques (S. Yla-Herttuala et al., Proc. Natl. Acad. Sci. USA 1991,88(12), 5252-5256). By inhibiting NF-κB activation and MCP-1 expression,triflusal and HTB may be of particular utility in the treatment orprevention of MCP-1-mediated disorders such as those mentioned above.

[0182] The results from example 9 show that triflusal and HTB inhibitalso the expression of TNF-α, which is regulated at transcriptionallevel, at least partially, by NF-κB (J. Yao et al, J. Biol. Chem. 1997,272(28), 17795-17801). It has been described that an excessive orunregulated TNF-α production is involved in a broad range of disorderssuch as rheumatoid arthritis, rheumatoid spondylitis, gouty arthritisand other arthritic conditions, arthrosis, sepsis, septic shock,endotoxic shock, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoidosis, pulmonary fibrosis, hepatitis,osteoporosis and other bone resorption disorders, reperfusion injury,transplant rejection, multiple sclerosis, lupus, fever and myalgias dueto infections, cachexia, acquired immune deficiency syndrome (AIDS),inflammatory bowel disease and pyresis (L. Sekut and K. M. Connolly,Drug News Perspect. 1996, 9(5), 261-269). By inhibiting NF-κB activationand TNF-α expression, triflusal and HTB may be of particular utility inthe treatment or prevention of TNF-α-mediated disorders such as thosementioned above.

[0183] The results from examples 11, 12 and 13 further show theusefulness of triflusal and HTB for the treatment or prevention ofneurodegenerative diseases, arthritis and cancer, respectively.

[0184] The concentrations at which effects are observed in theexperiments described in examples 1 to 13 are reached at the therapeuticdoses of triflusal commonly used in humans by the oral route.

[0185] Without wishing to be bound by what is herein stated, it isbelieved that the inhibition of the expression of proteins such asVCAM-1, iNOS, COX-2, MCP-1 and TNF-α by triflusal and HTB is mediated,at least partially, by an inhibition of the activation of thetranscription factor NF-κB. This notwithstanding, it is known that theexpression of the genes that encode these proteins may be activated byother agents. Since we have shown that triflusal and HTB inhibit theexpression of these genes (both in vitro and in vivo, in the case ofCOX-2), both products may also be useful in the treatment or preventionof disorders where there is an elevated expression of these genes thatis independent of NF-κB, which is also encompassed by the scope of thepresent invention.

1. A method for the treatment or prevention of a neurodegenerativedisease in a warm-blooded animal in need thereof, comprisingadministering to said animal an effective amount of a compound offormula I

wherein R represents hydrogen or COCH₃, or a pharmaceutically acceptablesalt or a prodrug thereof.
 2. The method of claim 1, where theneurodegenerative disease is dementia.
 3. The method of claim 1, whereinthe dementia comprises Alzheimer's disease, dementia followinginfection, or a combination thereof.
 4. The method of claim 1, whereinthe neurodegenerative disease is progressive ataxias.
 5. A method forthe treatment or prevention of Alzheimers disease in a human in needthereof which comprises administering to said human an effective amountof a compound of formula I

wherein R represents hydrogen or COCH₃, or a pharmaceutically acceptablesalt or prodrug thereof.